Showing papers on "Upstream activating sequence published in 2005"
TL;DR: Mediator was discovered because of its activity in a yeast RNA polymerase II (pol II) transcription system - it is needed for the system to respond to a transcriptional activator.
570 citations
TL;DR: Data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.
Abstract: Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb -14 site confers lineage-specific reporter gene expression in vivo. This kb -14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.
176 citations
TL;DR: It is demonstrated that a high density of RNA polymerase (Pol) is insufficient to elicit histone displacement, which suggests that histone eviction is modulated by factors that are not linked to elongating Pol II, and suggests that Histone depletion plays a causal role in mediating vigorous transcription in vivo.
Abstract: We show that histone-DNA interactions are disrupted across entire yeast heat shock genes upon their transcriptional activation. At HSP82, nucleosomal disassembly spans a domain of 3 kb, beginning upstream of the promoter and extending through the transcribed region. A kinetic analysis reveals that histone H4 loses contact with DNA within 45 s of thermal upshift. Nucleosomal reassembly, prompted by temperature downshift, is also rapid, detectable within 60 s. Prior to their eviction, promoter-associated histones are transiently hyperacetylated, while those in the coding region are not. An upstream activation sequence mutation that weakens the binding of heat shock factor obviates domain-wide remodeling, while deletion of the TATA box that nearly abolishes transcription is permissive to 5-end remodeling. The Swi/Snf complex is rapidly recruited to HSP82 upon heat shock. Nonetheless, domain-wide remodeling occurs efficiently in Swi/Snf mutants despite a sixfold reduction in transcription; it is also seen in gcn5, set1, and paf1 mutants. Contrary to current models, we demonstrate that a high density of RNA polymerase (Pol) is insufficient to elicit histone displacement. This finding suggests that histone eviction is modulated by factors that are not linked to elongating Pol II. It further suggests that histone depletion plays a causal role in mediating vigorous transcription in vivo and is not merely a consequence of it.
149 citations
TL;DR: Gal4 has been shown to activate the GAL1/10 upstream activation sequence upon Galactose catabolism as mentioned in this paper, which is the first detectable factor to be recruited to GAL 1/10 downstream activation sequence.
Abstract: Transcription activators are necessary for the precise regulation of gene expression in response to a variety of cellular signals. Many eukaryotic activators directly or indirectly facilitate recruitment of the transcription machinery to a promoter by enhancing the binding of chromatin remodeling factors, by counteracting the action of specific repressors, and/or by directly interacting with the transcription machinery (23, 26, 41). Acidic activating regions are common in eukaryotes and typically contain one or more critical hydrophobic residues and an abundance of acidic side chains. Strong acidic activators can be as short as 30 residues and are relatively insensitive to mutagenesis, and truncations of the activation regions result in a progressive loss of activity (20, 28). These results suggest that acidic activators do not fold into a typical structured protein domain. In agreement with this proposal, structural studies of the activators c-myc, CREB, and VP16 demonstrated that these activating regions are unstructured in the absence of an interacting partner (30, 42, 47). Sequence comparisons, the isolation of activators from random sequences, mutagenesis, and selection for mutations which increase function have not revealed any obvious similarity among the primary sequences of acidic activators, apart from being rich in acidic and hydrophobic residues (13, 19, 28). A central question in gene regulation is how these diverse activators function to stimulate transcription through the common set of factors comprising the transcription machinery.
Yeast Gal4, one of the first known acidic activators, is responsible for the regulation of genes involved in galactose catabolism (41). In the absence of galactose, Gal4 is inhibited by the repressor Gal80. Upon the addition of galactose, Gal4 is released from this repression by the activation of Gal3, which blocks the repressive function of Gal80 (27). Gal4 contains an N-terminal DNA binding and dimerization domain (residues 1 to 147), an N-terminal activating region (residues 148 to 196), and a C-terminal activating region (residues 768 to 881) (28). The most acidic part of the C-terminal activating region (residues 840 to 881) is the minimal segment required for strong activator function in vivo (28). The Gal4 activator functions to stimulate transcription in all eukaryotes tested, from yeast to humans, and activates transcription when fused to a heterologous DNA binding domain (5, 41). The Gal4 DNA binding domain has no activation activity when separated from the activating regions, although mutations within this domain have been reported to reduce activation in vivo (12).
Many direct activation targets of Gal4 have been proposed, based on a variety of biochemical, genetic, and molecular studies. In vivo assays have demonstrated that the acetyltransferase/coactivator complex SAGA, which is required for Gal4 induction, is the first detectable factor to be recruited to the GAL1/10 upstream activation sequence upon galactose induction (3, 7). In agreement with these studies, in vivo fluorescent resonance energy transfer (FRET) assays suggest that Gal4 and the SAGA subunit Tra1 are in close proximity only after induction (4). In vitro protein-protein interaction studies have demonstrated binding of Gal4 to TATA-binding protein (TBP), TFIIB, Swi/Snf, Mediator, and SAGA (1, 22, 37, 51). In Mediator, Gal4 binds to the Srb10 and Gal11 subunits, binding Gal11 in two separate regions. Other acidic activators such as yeast Gcn4 have also been found to interact with numerous polypeptides in vitro (6, 15, 35). The short nonconserved sequences of acidic activators, coupled with the observed in vitro binding of many polypeptides, raise the question of how these factors specifically recognize their relevant targets. Nearly every general transcription factor and coactivator complex has been proposed as a direct activator target, but in only a few cases has the functional relevance of these interactions been demonstrated (4, 6, 16, 18, 39, 49). One limitation of many previous studies is that the activator targets were not defined in functional transcription complexes, but instead were identified using isolated factors or individual subunits of large complexes.
For this work, we used site-specific photo-cross-linkers inserted within the Gal4 C-terminal activating region to identify polypeptides in close proximity to the activator while the activator stimulates transcription. This approach revealed six polypeptides that cross-link to the activating region. Three of these polypeptides (Tra1, Gal11, and Taf12) are subunits of four complexes previously implicated in gene regulation, namely, Mediator, SAGA, NuA4, and TFIID. Remarkably, these three cross-linking targets were also identified in a cross-linking assay with the acidic activator Gcn4 (17). Thus, two activators with unrelated sequences interact with the same set of three targets during transcription activation. Unexpectedly, we found that Ste12, which is itself a gene-specific transcription factor, is also a specific target of Gal4. Functional studies demonstrate that these Gal4 cross-linking targets make differential contributions to activation by Gal4.
131 citations
TL;DR: By elucidating the function of one of the previously unrecognized, putative transcription units, this work provides evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.
Abstract: By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.
104 citations
TL;DR: It is suggested that sequence-independent upstream DNA interactions with alphaCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the alpha-binding sites.
Abstract: The C-terminal domains of the two α-subunits (αCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–αCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–αCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of αCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of αCTD decreased the composite second-order association rate constant, ka, of RNAP for the lacUV5 promoter by ≈10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k2. Removal of promoter DNA upstream of the -35 element also decreased both ka and k2 ≈10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments. The αCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with αCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the α-binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–αCTD interactions play an even larger role in transcription initiation than appreciated previously.
81 citations
TL;DR: In this paper, the authors used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv).
Abstract: We used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv. Nipponbare). Our T-DNA enhancer trapping cassette consisted of two principle components: (1) the minimal promoter-equipped gal4 gene placed adjacent to the right border, and (2) the green fluorescent protein gene (gfp) fused to the upstream activation sequence element (UAS) to which GAL4 binds and activates expression, so that gfp expression corresponds to gal4 expression. Agrobacterium-mediated integration of the cassette into the rice genome often brings the gal4 gene under transcriptional control of local genomic enhancers and promoters, resulting in gal4/gfp expression patterns ranging in specificity from single-cell types to constitutive expression. We produced more than 13 000 enhancer trap lines with this cassette and screened T(0) adult plants (1982 lines), T(1) seed (2684 lines) and T(1) seedlings (2667 lines) for gfp expression. Approximately 30% of the lines produced GFP, and we identified lines with gfp expression in specific cell types of all major organs of the rice plant. Subsequently, using the GUS reporter gene (uidA), we demonstrated that UAS:geneX constructs can be transactivated in specific cell types where gal4 and gfp are expressed, thus providing an excellent system for the manipulation of gene expression and physiological function in specific cell types of rice.
74 citations
TL;DR: An activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence is proposed, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter.
Abstract: Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.
72 citations
TL;DR: It is shown that normal LHX4 binds to a human-specific element and subsequently activates transcription from the proximal upstream regulatory sequence of POUIF1, a gene encoding a POU homeodomain transcription factor known as the main regulator of GH expression that is consistent with the existence of an LHx4-driven pathway leading to the expression of GH through transcriptional activation of Pou1F1.
Abstract: Context: Pituitary development depends on the actions of a large number of transcription factors. Among them, LHX4 is believed to play a crucial role, as suggested by the dominantly inherited GH deficiency associated with the recently identified LHX4 mutation, although the precise mechanism underlying this phenotype is still to be elucidated. Objective: The objective of this study was to gain insight into both the function of LHX4 and the pathophysiology of the LHX4-related syndrome. We sought potential targets of this factor and assessed the abilities of various recombinant LHX4 isoforms expressed in Chinese hamster ovary cells to bind to and activate the POUI1F1 upstream regulatory sequence. Results: We show that normal LHX4 binds to a human-specific element and subsequently activates transcription from the proximal upstream regulatory sequence of POUIF1, a gene encoding a POU homeodomain transcription factor known as the main regulator of GH expression. As shown in this cell system, the mutant LHX4 pro...
58 citations
TL;DR: Results provide strong evidence that TFIIS plays an important role in the initiation of transcription at GAL1 in addition to its well-characterized roles in transcription elongation.
Abstract: TFIIS is a transcription elongation factor that has been extensively studied biochemically. Although the in vitro mechanisms by which TFIIS stimulates RNA transcript cleavage and polymerase read-through have been well characterized, its in vivo roles remain unclear. To better understand TFIIS function in vivo, we have examined its role during Gal4-mediated activation of the Saccharomyces cerevisiae GAL1 gene. Surprisingly, TFIIS is strongly associated with the GAL1 upstream activating sequence. In addition, TFIIS recruitment to Gal4-binding sites is dependent on Gal4, SAGA, and Mediator but not on RNA polymerase II (Pol II). The association of TFIIS is also necessary for the optimal recruitment of TATA-binding protein and Pol II to the GAL1 promoter. These results provide strong evidence that TFIIS plays an important role in the initiation of transcription at GAL1 in addition to its well-characterized roles in transcription elongation.
58 citations
TL;DR: It is demonstrated that dMnt is a transcriptional repressor that regulates D. melanogaster body size and generated a dMNT null allele, which results in flies with larger cells, increased weight, and decreased life span compared to wild-type flies.
Abstract: The Myc-Max-Mad/Mnt network of transcription factors has been implicated in oncogenesis and the regulation of proliferation in vertebrate cells. The identification of Myc and Max homologs in Drosophila melanogaster has demonstrated a critical role for dMyc in cell growth control. In this report, we identify and characterize the third member of this network, dMnt, the sole fly homolog of the mammalian Mnt and Mad family of transcriptional repressors. dMnt possesses two regions characteristic of Mad and Mnt proteins: a basic helix-loop-helix-zipper domain, through which it dimerizes with dMax to form a sequence-specific DNA binding complex, and a Sin-interacting domain, which mediates interaction with the dSin3 corepressor. Using the upstream activation sequence/GAL4 system, we show that expression of dMnt results in an inhibition of cellular growth and proliferation. Furthermore, we have generated a dMnt null allele, which results in flies with larger cells, increased weight, and decreased life span compared to wild-type flies. Our results demonstrate that dMnt is a transcriptional repressor that regulates D. melanogaster body size.
TL;DR: It is proposed that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase.
Abstract: Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)–promoter complex and/or the rate of subsequent conformational changes required to form the open complex (RPO). Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble. Kinetic studies with the λPR promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair -47 greatly increases the rate of forming RPO, without significantly affecting its rate of dissociation. We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RPO formation, a step that occurs after polymerase binds. Footprinting experiments reveal striking structural differences downstream of the transcription start site (+1) in the first kinetically significant intermediate when upstream DNA is present. On the template strand, the DNase I downstream boundary of this early intermediate is +20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond -47 is removed. KMnO4 footprinting reveals an identical initiation bubble (-11 to +2), but unusual reactivity of template strand upstream cytosines (-12, -14, and -15) on the truncated promoter. Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase.
TL;DR: It is found that TFII-I is capable of acting at both basal and regulatory sites in one promoter and that the human VEGFR-2 promoter is functionally counter-regulated by TF II-I andTFII-IRD1.
TL;DR: It is proposed that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the Efficiency of promoter clearance.
TL;DR: A functional dissection of the highly constrained architecture of the rb2 promoter shows that a ‘one‐site’ minimal UAS suffices for activation by Ptr2, and specifies the required placement of this site.
Abstract: The archaeal transcription apparatus is chimeric: its core components (RNA polymerase and basal factors) closely resemble those of eukaryotic RNA polymerase II, but the putative archaeal transcriptional regulators are overwhelmingly of bacterial type. Particular interest attaches to how these bacterial-type effectors, especially activators, regulate a eukaryote-like transcription system. The hyperthermophilic archaeon Methanocaldococcus jannaschii encodes a potent transcriptional activator, Ptr2, related to the Lrp/AsnC family of bacterial regulators. Ptr2 activates rubredoxin 2 (rb2) transcription through a bipartite upstream activating site (UAS), and conveys its stimulatory effects on its cognate transcription machinery through direct recruitment of the TATA binding protein (TBP). A functional dissection of the highly constrained architecture of the rb2 promoter shows that a 'one-site' minimal UAS suffices for activation by Ptr2, and specifies the required placement of this site. The presence of such a simplified UAS upstream of the natural rubrerythrin (rbr) promoter also suffices for positive regulation by Ptr2 in vitro, and TBP recruitment remains the primary means of transcriptional activation at this promoter.
TL;DR: The hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis‐regulatory element that regulates the transcription of the h Oct4 promoter by the binding of Sp family transcription factors.
Abstract: Octamer-binding transcription factor-4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5' upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5' upstream region and identified cis-regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis-regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis-acting element located between CR1 and CR2. In addition, the sequence -380/-1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the -380/-1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis-regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.
TL;DR: Analysis of a zrt1Δ zrt2Δ mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase, and this work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.
TL;DR: It is concluded that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGF BP-3, and hypermethylation of these sequences selectively suppresses p 53 induced IGFBP -3 expression in HepG2 cells.
Abstract: Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells.
TL;DR: It is reported here that activation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-I increases transcription from the ribosomal DNA promoter in both myeloid cells and mouse fibroblasts, providing one explanation for the reported effects of the IGF/IRS-1 axis on cell and body size in animals and cells in culture.
TL;DR: The Gal4 trap system described here uses the 5XUAS-LUC and 5X UAS rsGFP-GUS as reporters and allows for in planta quantitative screening, including the rapid monitoring for silencing.
Abstract: Background
Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study.
TL;DR: The conformation of all seven DNA elements located upstream of the major E. coli rRNA P1 promoters is analyzed, showing that all seven rRNA upstream sequences are intrinsically curved.
Abstract: Ribosomal RNAs in E. coli are transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the major E. coli rRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for the rrnB and rrnD fragments and less pronounced for the rrnA and rrnE operons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.
TL;DR: The findings show that expression of C/EBPbeta, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.
Abstract: For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPbeta (CCAAT/enhancer-binding protein beta) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPbeta genomic fragments containing the 8451 bp 5' upstream of the transcription start site did not contain amino-acid-responsive elements. However, deletion analysis of the genomic region located 3' downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acid-responsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPbeta enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPbeta promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPbeta itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPbeta, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.
TL;DR: In this paper, a putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website.
Abstract: A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the ΔYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.
TL;DR: A novel coactivator function for Cyc8p/Tup1p at the level of activator binding is revealed and it is suggested that Gcn4p may enhance its own binding to the upstream activation sequence (UAS).
Abstract: The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Δ cells and to a lesser extent in tup1Δ cells. The defects in Gcn4p binding and transcriptional activation in cyc8Δ cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Δ cells. The impairment of Gcn4p binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3′ ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.
TL;DR: Data suggest that the human proximal cGMP-dependent protein kinase I promoter is regulated by tandem E-boxes that bind upstream stimulatory factors.
TL;DR: It is shown that promoters can also interact positively, and transcriptional elongation over this class of intragenic promoters will generate co‐regulated sense–antisense transcripts, or, alternatively initiating transcripts, thus expanding the diversity and complexity of the human transcriptome.
Abstract: Transcriptional interference denotes negative cis effects between promoters. Here, we show that promoters can also interact positively. Bidirectional RNA polymerase II (Pol II) elongation over the silent human endogenous retrovirus (HERV)-K 18 promoter (representative of 2.5 x 10(3) similar promoters genomewide) activates transcription. In tandem constructs, an upstream promoter activates HERV-K 18 transcription. This is abolished by inversion of the upstream promoter, or by insertion of a poly(A) signal between the promoters; transcription is restored by poly(A) signal mutants. TATA-box mutants in the upstream promoter reduce HERV-K 18 transcription. Experiments with the same promoters in a convergent orientation produce similar effects. A small promoter deletion partially restores HERV-K 18 activity, consistent with activation resulting from repressor repulsion by the elongating Pol II. Transcriptional elongation over this class of intragenic promoters will generate co-regulated sense-antisense transcripts, or, alternatively initiating transcripts, thus expanding the diversity and complexity of the human transcriptome.
TL;DR: A powerful method is developed that uses a positive feedback mechanism to amplify the activities of individual IRES elements in mammalian cells and demonstrates the utility of this vector system for the screening, identification, and analysis ofIRES elements.
Abstract: In earlier studies, we identified short (6- to 22-nt) sequences that functioned as internal ribosome entry sites (IRESes) and enhanced translation. The size of these IRES elements suggested that they might be prevalent within the messenger population and that individual elements might affect the translation of different groups of mRNAs. To begin to assess the number of different IRES elements in mammalian cells, we have developed a powerful method that uses a positive feedback mechanism to amplify the activities of individual IRES elements. This method uses a vector that encodes a dicistronic mRNA with a reporter gene (Renilla luciferase or the EGFP) as the first cistron and the yeast Gal4/viral protein 16 (VP16) transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing four copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4/VP16, which binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4/VP16. A corresponding increase in the translation of the first cistron (luciferase or EGFP) is monitored either by measuring luciferase activity or by using FACS. The latter enables IRES-positive cells to be isolated. We present tests of the feedback mechanism by using an IRES module from Gtx homeodomain mRNA and an IRES from hepatitis C virus and demonstrate the utility of this vector system for the screening, identification, and analysis of IRES elements.
TL;DR: It is demonstrated that hairpin-TFO can be employed in vivo for targeted activation of gene expression of two genes mapping on chromosome XI of Saccharomyces cerevisiae.
Abstract: Triplex forming oligonucleotides (TFO) are known to be potential agents for modifying gene function. In most instances they are utilized for repression of transcription. However hybrid molecules containing cis-acting elements in a duplex DNA in a hairpin form contiguously with the TFO can bind transcription factors in vitro. In the present manuscript we demonstrate that hairpin-TFO can be employed in vivo for targeted activation of gene expression of two genes mapping on chromosome XI of Saccharomyces cerevisiae. The cis-acting GAL4 protein-binding site contained in the hairpin-TFO is targeted in vivo to the 5′ upstream sequence of STE6 and CBT1 genes that are transcribed in opposite directions and share a poly(pu/py) sequence that can form triple helical structure. The hairpin-TFO is targeted to this site and promotes the activation of both the genes. These results demonstrate four important aspects relating to activation of gene expression: (i) accessibility of duplex DNA packaged into chromatin to triplex forming sequences in vivo, (ii) the potential use of hairpin-TFO in therapeutics by activation of transcription in vivo, (iii) Sharing of transcription factors between two genes transcribed in opposite directions and (iv) specific activation of genes even when their cognate site is not covalently linked to the gene being activated. (Mol Cell Biochem 278: 147–155, 2005)
TL;DR: Results indicate that the promoter sequence spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions, and is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant.
Abstract: The sequences surrounding the -12/-24 motif of the m-xylene-responsive sigma54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH sigma54 promoter variants known to have various degrees of efficacy and affinity for sigma54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the -12/-24 region of the wild-type PnifH and its higher sigma54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.
TL;DR: Depletion of zinc from the growth medium of wild-type cells results in alterations in phospholipid composition including an increase in PI (phosphatidylinositol) and a decrease in phosphatidylethanolamine and a reduction in PS synthase in response to zinc depletion.
Abstract: The yeast Saccharomyces cerevisiae has the ability to cope with a variety of stress conditions (e.g. zinc deficiency) by regulating the expression of enzyme activities including those involved with phospholipid synthesis. Zinc is an essential mineral required for the growth and metabolism of S. cerevisiae . Depletion of zinc from the growth medium of wild-type cells results in alterations in phospholipid composition including an increase in PI (phosphatidylinositol) and a decrease in phosphatidylethanolamine. These changes can be attributed to an increase in PIS1 -encoded PI synthase activity and a decrease in the activities of several CDP-diacylglycerol pathway enzymes including the CHO1 -encoded PS (phosphatidylserine) synthase. The reduction in PS synthase in response to zinc depletion is due to a repression mechanism that involves the UAS INO (inositol upstream activating sequence) element in the CHO1 promoter and the negative transcription factor Opi1p. These factors are also responsible for the inositol-mediated repression of CHO1 . This regulation may play an important role in allowing cells to adapt to zinc deficiency given the essential roles that phospholipids play in the structure and function of cellular membranes.